The present invention is directed to a method of producing spider dragline and/or flagelliform proteins. The invention is further directed to a method of producing spider threads and to a dragline/flagelliform protein or dragline/flagelliform protein thread produced by these methods. The invention further provides the use of these proteins/threads in the field of biotechnology and/or medicine, in particular in the manufacture of wound closure or coverage systems, suture materials and in the manufacture of replacement materials, preferably artificial cartilage or tendon materials, as well as in other commercial applications.
Spider dragline silk has extraordinary properties (1) originating in its composition as a semi crystalline polymer (2) that contains crystalline regions embedded in an amorphous matrix. X-ray diffraction and NMR show the crystalline regions to consist of pleated beta sheets of polyalanine stretches which are giving strength to the thread (3,4), while the predominant secondary structure of the amorphous matrix is a glycine rich 31 helix providing elasticity (5). Freshly secreted silk proteins are stored at high concentrations (6) as a liquid crystalline dope (7,8) that is altered by changes in ionic composition, pH (from pH 6.9 to 6.3) (9,10) and water extraction (10,11) during its passage through the spinning duct to be finally converted into a solid thread induced by extensional flow (12).
All dragline silks studied so far consist of at least two different proteins with molecular masses of up to several hundred kDa (13). The individual contribution of the two major dragline silk proteins of Araneus diadematus, ADF-3 and ADF-4, to dragline thread assembly and structure has not been determined so far. Analyzing the primary structures revealed that ADF-3 and ADF-4 (14,15) have similar proline contents and polyalanine motifs, but they differ in glutamine and serine content as well as in length of the glycine-rich regions. Importantly, the properties of silk threads cannot be inferred from the underlying protein sequences. Although the quality of a silk thread is based on the primary structure of the involved proteins, it further depends on the silk assembly process (8), which necessitates experimental analysis of structural and assembly properties.
Scientific and commercial interest initiated the investigation of industrial scale manufacturing of spider silk. Native spider silk production is impractical due to the cannibalism of spiders, and artificial production has encountered problems in achieving both sufficient protein yield and quality thread-assembly. Bacterial expression yielded low protein levels (16), likely caused by a different codon usage in bacteria and in spiders. Synthetic genes with a codon usage adapted to the expression host led to higher yields (13,17), but the proteins synthesized thereof showed different characteristics in comparison to native spider silks. Expression of partial dragline silk cDNAs in mammalian cell lines did yield silk proteins (e.g. ADF-3) that could be artificially spun into ‘silken’ threads, albeit as yet of inferior quality (18).
WO03060099 relates to methods and devices for spinning biofilament proteins into fibers. This invention is particularly useful for spinning recombinant silk proteins from aqueous solutions and enhancing the strength of the fibers and practicality of manufacture such as to render commercial production and use of such fibers practicable. Therein, it is disclosed to express spider silk proteins in mammalian cells, e.g. transgenic goat mammary gland cells.